Erythromelalgic Microvascular Disturbances, Major Thrombosis

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Review Article Open Access Erythromelalgic Microvascular Disturbances, Major Thrombosis and Hemorrhagic Manifestations of Thrombocythemia in Patients with Essential Thrombocythemia and Polycythemia Vera: Therapeutic Implications Jan Jacques Michiels* Academic Hospital Dijkzigt, Erasmus University Rotterdam (EUR 1974-1998), Department of Hematology, Antwerp University Hospital, Belgium, and Goodheart Institute, Bloodcoagulation and Vascular Medicine Center, Rotterdam, (1998-2014) The Netherlands

Abstract Aspirin responsive erythromelalgia is the presenting symptom of thrombocythemia in patients with essential thrombocythemia (ET) and polycythemia vera (PV). Skin punch biopsies taken from the affected areas of erythromelalgia and acrocyanotic compications show typical arteriolar inflammation, fibromuscular intimal proliferation and thrombotic occlusions. If left untreated both microvascular and major thrombosis frequently do occur in thrombocythemia in ET and PV patients, but can easily be cured and prevented by low dose aspirin. The stratification as low, intermediate and high thrombotic risk in the retrospective Bergamo studies has been performed in ET patients not treated with aspirin. The risk of thrombosis in aspirin treated ET and PV is not age dependent when on low dose aspirin, and does recur when not on low dose aspirin during follow-up. The persistence of the Bergamo definition of low, intermediate and high thrombotic risk in the 2012 International Prognostic Score of Thrombosis in ET (IPSET) is applicable for JAK2 mutated ET and PV patients not on aspirin and has led to significant overtreatment with hydroxyurea. MPN disease burden in patients with JAK2V617F positive ET and PV is related to JAK2 allele burden and associated leukocytosis, thrombocytosis, constitutional symptoms and splenomegaly. Low dose peglyated interferon (PegasysR, 45 µg/ ml once per week or every two weeks) is becoming the first line myeloreductive treatment option to postpone the useof hydroxyurea as long as possible. Von Willebrand factor (VWF) mediated platelet thrombi formation, as well as increased proteolysis of the VWF multimers in one and the same patient do occur simultaneously or in sequence leading to the paradoxical occurrence of thrombosis at platelet count aboe 400x109/L and bleeding at platelet counts above 1000x109/L due to an acquired von Willebrand disease type 2A.

Keywords: Erythromelalgia; Hemorrhages; Arterial thrombosis; Prospective Clinical Studies on Erythromelalgia and Thrombocythemia; Polycythemia vera; Essential thrombocythemia; Acrocyanotic Toes or Fingers in ET and PV Hemorrhagic thrombocythemia; Thrombotic thrombocythemia; Aspirin; Myeloproliferative disorders The broad spectrum of clinical features of erythromelalgia and peripheral, cerebral and coronary ischemic events in 24 patients with Introduction myeloproliferative thrombocythemia (11 ET and 13 PVT patients were collected by the Rotterdam MPN Study Group in the period Mitchel first reported a syndrome of redness and burning pain in between 1975 and 1981 (Table 1) [3,4]. Complaints of erythromelalgia the extremities, which were labeled erythromelalgia [1]. The relief of consisted of painful red to cyanotic toes or fingers. The pain may vary pain for several days after a single dose of aspirin (500 mg) is specific from prickling, pins and needle stick sensation to a severe burning and can be used as a diagnostic criterion for erythromelalgia [2]. We pain. The involved areas of fore foot toe or fingers are always swollen discovered that aspirin responsive erythromelalgia is causally related to and painful on palpation and touch (Figures 1 and 2) [3-6]. At time thrombocythemia in patients with essential thrombocythemia (ET) and of burning pain severe attacks of tingling prickling may occur [3,4]. polycythemia vera (PV) at platelet counts around and above 400x10/L The burning pain of erythromelalgia increased on walking compelling [3,4]. By demonstratin that erythromelalgia in thrombocythemia the patient to rest with the involved extremity elevated. After rest with disappeared for 2 to 4 days after a single dose of aspirin (500 mg). This the involved extremity elevated, the erythromelalgic redness may could be attributed to the irreversible inactivation of platelet cyco- disappear but the extremity remains swollen, acrocyanotic and painful oxygenase activity for 2 to 4 days by acetyl-salicylic acid (aspirin). on touch. Fingertips or toes remain red, swollen and severely painful As sodium salicylic acide has no cyclo-oxygenase inhibiting acticity like a burn. Acrocyanotic spots in erythromelalgic areas were present in on platelets and has no effect at all on erythromelalgia we concluded 8 cases indicating localized ischemia (Table 1) [3-6]. In one ET patient in 1985 that aspirin responsive erythromelalgia in thrombocythemia is caused by platelet mediated microvascular infammatrory and thrombosis in ET and PV patients [3-6]. *Corresponding author: Jan Jacques Michiels, Goodheart Institute, The present study reviews the original description of the Bloodcoagulation and Vascular Medicine Center, Erasmus Tower, Veenmos 13, 3069 broad spectrum of microvascular, thrombotic and hemorrhagic AT Rotterdam, Netherlands, Tel: +316-26970534; Email: [email protected] manifestations in ET and PV patients related to peripheral blood Received April 17, 2013; Accepted July 07, 2014; Published July 15, 2014 cell counts, bone marrow features, and burden of myelopoliferative Citation: Michiels JJ (2014) Erythromelalgic Microvascular Disturbances, Major neoplasm (MNP) disease.The pathophysiology of platelet-mediated Thrombosis and Hemorrhagic Manifestations of Thrombocythemia in Patients with arterial thrombosis and bleeding in ET and PV patients is discussed Essential Thrombocythemia and Polycythemia Vera: Therapeutic Implications. J Hematol Thromb Dis 2: 149. doi: 10.4172/2329-8790.1000149 and interpreted in view of the recent literature and updated treatment recommendations for the prevention of platelet-mediated thrombosis Copyright: © 2014 Michiels JJ. This is an open-access article distributed under and bleeding in thrombocythemia and by reduction of MPN disease the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and burden by pegylated interferon or hydroxyurea. source are credited.

J Hematol Thromb Dis ISSN: 2329-8790 JHTD, an open access journal Volume 2 • Issue 5 • 1000149 Citation: Michiels JJ (2014) Erythromelalgic Microvascular Disturbances, Major Thrombosis and Hemorrhagic Manifestations of Thrombocythemia in Patients with Essential Thrombocythemia and Polycythemia Vera: Therapeutic Implications. J Hematol Thromb Dis 2: 149. doi: 10.4172/2329- 8790.1000149

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Number Gender Platelets/ Hb/Ht Erythromelalgia TIA/ACS History ETT Diagnosis Age/yrs 109/L mmol/L % Toes Fingers Skin Gangrene months A1 ET F 62 792 10.3/50 bilateral present 45 A2 ET M 39 887 10.0/51 bilateral - - present TIA/ACS 154 A3 ET F 42 911 8.9/47 - unilateral yes ACS 60 A4 ET M 47 614 8.0/39 unilateral - - present TIA 12 A5 ET M 46 939 8.3/40 unilateral - - present 4 A6 ET M 33 742 9.8/45 - - - - TIA/ACS 20 A7 ET M 45 567 9.5/46 bilateral yes ACS 60 A8 ET M 39 875 8.8/43 bilateral bilateral yes - - 30 A9 ET M 51 690 8.8/45 bilateral - - present - 20 A10 ET M 40 1440 8.6/43 unilateral - - present - 30 A11 ET F 72 1435 9.4/46 unilateral - - - - 30 A12 PVT M 63 1932 11.1/56 bilateral - - - - 24 A13 PVT F 75 1800 12.1/62 - unilateral yes - - 3 A14 PVT M 61 952 8.3/45 bilateral - - - - 0 A15 PVT M 53 636 7.7/39 unilateral - - present - 36 A16 PVT F 60 1065 13.4/68 bilateral - - - - 48 A17 PVT F 49 728 10.9/57 - bilateral - - - 1 A18 PVT M 66 1035 12.2/64 bilateral - - - - 18 A19 PVT M 71 1320 13.3/70 unilateral - - - - 2 A20 PVT M 65 1300 11.9/65 unilateral - - present - 24 A21 PVT F 55 1085 12.2/61 - unilateral - - - 4 A22 PVT F 59 708 11.0/59 - bilateral yes - - 3 A23 PVT F 74 959 13.1/72 unilateral unilateral - - - 24 A24 PVT M 71 609 12.5/66 unilateral 6

Table 1: Erythromelalgia and microvascular disturbances in 24 patients with ETT (11 ET and 13 PV) [3].

the fingertipss was followed by cold, swollen acrocyanosis of 4 fingers of the left hand (aspirin responsive blue finger and black toe syndrome in PVT, Figure 1) [1-4]. In ET erythromelalgia already develops at pflaftefleft counft fin excess off 350 fto9 400x10 /L. WThen sympftomaftfic at ‘normal’ platelet count around 350x109/L in PV in remission by phlebotomy alone platelet count usually rise to values above 400x109/L during aspirin treatment [3,4]. The time lapse between first complaints of typical erythromelalgia and the diagnosis of ET or PV elapsed from a few months to 5 years, (mean 2,25) years [3,4]. One ET patients (A2) had a very long history of erythromelalgia complicated by peripheral gangrene, atypical TIAs and recurrent coronary ischemic events. Seven cases with longstanding erythromelalgic signs and symptoms developed ischemic complications: acrocyanosis with blister formation of the big toe (A1), a necrotic spot in the tip of a toe in 4 (A1, A1, A4, A20) [3,4]. In PV case 20 with red congestion of the big and second toe and a necrotic spot of the third toe bone marrow biopsy from the anterior iliac crest was complicated by wound dehiscent and subcutaneous ecchymosis. Sympathectomy because of ischemic complication was not effective in 5 cases with blue black toes and digital gangrene (thrombo- angiitis obliterans) in the absence of infection and inflammation in 3 Figure 1: A. Severe burning painand red, warm congestion of big and litlle (A4, A5, A10), and necrosis of the distal phalanx of a toe in 2 (A2, toes, sole of forefoot, and lateral edge of right foot are evident 1 week after A9). Angiogram in one ET case (A1) with erythromelalgia complicated discontinuation of aspirin. B. Burning, aching pain and mottled red-blue discoloration of sole of right by digital gangrene showed abrupt occlusions of toe arteries without forefoot are present 2 weeks after discontinuation of aspirin. any evidence of vascular pathology of the supplying arteries [5,6]. The C. Progression of erythromelalgia to painfull swollen cyanotic fingers and sequential occurrence of aspirin responsive erythromelalgic, ocular painful swelling with mottled redness of left hand palm. and cerebral microvascular disturbances in relation to platelet counts D. Arteriogram of left hand shown in c show absence of superficial arterial arcade and occulsion of digiital arteries except the thumb (arrows). and the effects of platelet reduction during 15 years follow-up has been describe in detail 6. Transient cerebral ischemic events in one PV patient (A24) was followed by overt erythromelalgia in the finger tips, which (A11), warm painful swollen redness of the big toe was accompanied were left untreated and complicated by acrocyanosis of 4 fingers of the by a cold dark blue cyanotic non-swollen third toe. In a third patient left hand showing abrupt arterial occlusions of the hand arcade and of (A24, PV) who presented with typical pronounced erythromelalgia of supplying digital finger arteries of the 4 involved acrocynotic swollen

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relief of erythromelagic and/or ischemic pain and restoration of the A B C ischemic acral circulation disturbances (thrombo-angiitis obliterans) in ET and PVT patients could be obtained by long-term treatment with low-dose aspirin (250 mg/day). In the five patients with erythromelalgia complicated by digital grangrene (Table 1) [3,4] experienced the favorable effect of aspirin, but it took about one or two weeks before all ischemic symptoms completely disappeared [5,6]. If left untreated,

D E F cold cyanotic fingertip or toe tips may develop, remain painful and resistant to aspirin very likely due to irreversible digital arteriolar occlusion. Standard therapy with oral anticoagulants (warfarin) and reduction of hematocrit to normal by bloodletting in PVT did not affect the erythromelalgic ischemic symptoms and acrocyanotic digital ischemia and gangrene (thrombo-angiitis obliterans).

Figure 2: A. burning pain and red congestion of forefoot sole and second and Histopathology of Erythromelalgia in Thrombocythemia third toes and bluish discoloration of very painful big toe of right foot (see also C). In the 1970s very little was known about the histopathology of B. Thermogram of right foot showing surface skin temperature with erythromelalgia in thrombocythemia as the opportunity to examine erythromelalgia exceding 310 Celsius (see also A and C). C. Detail of very painfull and bluish ischemic big toe with skin surface tissue from the involved extremities of typical fresh erythromelalgia temperature 50 Celsius lower compared to erythromelalgic forefoot sole in A and of its acrocyanotic complications was not or only scarcely available and B. [7]. We performed between 1975 and 1980 skin punch biopsies from D. Progression of untreated erythromelalgia into painful “black” big toe and erythromelalgic areas and from acrocyanotic areas in thrombocythemia peeling of the skin of second and third toes while oncoumarin treatment. E. Complete restoration of painful ischemic big toe and erythromelalgic second patients (ET and PV) [7]. The histological appearances of a skin and third toe within one week by treatment with aspirin 500 mg once daily. punch biopsy from anarea of recently relapsed erythromelalgia 1 F. Relapse of painful red blue toes did occur 2 weeks after discontinuation of week after discontinuation of aspirin, show arteriolar leasions without aspirin by the patient. After reinstitution the erythromelalgia disappeared again, which was interpreted by the patient as ‘The Miracle of Rotterdam” involvement of venules, capillaries of nerves (Figure 3) [7]. Arterioles showed pronounced proliferation and degeneration in their vessel walls. The lining endothelial cells are swollen some with large nuclei. The inner layer of the arteriolar vessel wall was thickened by intimal proliferation of cells with narrowing of the lumen. With factor VIII antisera an affected arteriole with intimal thickening shows an inner layer of fluorescencent endothelial cells (Figure 4) and with the antiserum against smooth muscle cells strong flurescence was seen in the proliferated subendothelial intimal cell zone (fibromuscular intimal proliferation, Figure 3) [7]. Slight intravascular and perivascular infiltration with inflammatory cells and some perivascular fibrosis is present. The internal elastic lamina is seen to be splitted by proliferating smooth muscle cells (fibromuscular intimal proliferation, Figure 3) [7]. The histopathological lesions from relapsed erythromelalgia complicated by bluish spots 3 weeks after discontinuation of aspirin, show arteriolar lesions of fibromuscalr intimal proliferation

Figure 3: Histology of erythromelalgia one week after discontinuation of aspirin showing arterioles with pronounced fibromuscular intima proliferation with swollen endothelial cells and muscle cell (Figure 4) proliferation of the inner part of media with associated degenerative vacuola cytolasmic swelling (upper panels and left middle). Fragmented internal elastic lamina (arrow) between proliferated smooth muscle cells in the van Gieson stain (right middle panel): fibromuscular intimal proliferation.Histology of erythromelalgia complicated by acrocyanotic ischemia 3 weeks after discontinuation of aspirin in essential thrombocythemia showing partial thrombotic occlusion of arteriole wit fibromuscular intimal proliferation (lower left). Occlusion of arteriole by organized thrombus with perivascular fibrosis (lower middle). Endstage vascular pathology of acrocyanotic erythromelalgia showing onionlike appearance of fibrosed occluded arteriole. Immunoflurescence of a biopsy from an erythromelalgic area with an arteriole occluded by a platelet rich thrombus with a strong staining for von Willebrand factor of the thrombus and of endothelial cells (central panel) [7]. .

Figure 4: Immunoflurescence of platelet-mediated arteriolar intima thickening fingers (Figure 1) [5,6]. Strikingly, the arterial pulses of arteriae dorsales from erythromelalgic area in essential thrombocythemia showing a single layer pedis and arteriae tibiales remain normal in all cases of erythromelalgia of endothelial cells with factor VIII antiserum (left) and of proliferated muscle cells with smooth muscle antiserum in an injured arteriole (right). complicated by peripheral acrocyanosis or digital gangrene. Complete

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screening with brain CT, carotid duplex, ultrasonography, or cardiac investigations did not reveal any obvious coexistent abnormality of cause of MIAs in affected ET and PV patients [9-11]. Three cases (1ET and 2 PV) developed necrotic toes and were diagnosed as thrombo-angiitis obliterans (Buerger’s disease) of the toes, which were already amputated by a surgeon before referral to us because of thrombocythemia for expert evaluation and treatment recommendation. The 1980 Rotterdam Clinical and Pathological Criteria Figure 5: Hematoxiline Esosin (H&E) stained sectionsfrom an acrocyanotic for ET and PV “black toe” with pregangrene diagnosedas Burger’s disease in an ET patient (A 25). A: Arteriole with organized thrombus within the lumen (H&E stan 300X). B: Focusing between 1975 and 1980 on the causal relation between Onionlike appearance of fibrosed wall of a small digital artery partially occluded erythromelalgia and thrombocythemia in ET and PV patients, we were by an organized thrombus with signs of recanalization. able to document the very early stage of ET by the use of the Rotterdam Clinical and Pathological (RCP) criteria for ET and PV (Table 2) [3,4]. 9 occluded by an organized fresh von willebrand factor rich platelet Since 1975 we found platelets in excess of 400 x 10 /L, and an increase of thrombi (Figure 3). The skin wedge excision from an acrocynotic clustered enlarged megakaryocytes in a bone marrow biopsy material erythromelalgic toe mainly shows arterioles with fibromuscular intimal diagnostic for ET (Table 2) [3,4]. Iron stain of bone marrow histology proliferation with narrowing of the lumen and on top of that occluded specimens from ET and PV patients were performed with Prussian arterioles by fresh organized thrombus is seen in erythromelalgic areas blue reagents. The RCP criteria of ET and PV were determined in complicated by acrocyanosis (Figures 3 and 5). The thickened arteriolar 1980 based on careful prospective documentation of peripheral blood microvasculature ultimately show occluded arterioles by thrombi with and bone marrow histology from bone marrow biopsy material. 9 perivascular fibrosis without inflammatory cell infiltration give rise to Platelets in excess of 400×10 /L, and an increase of clustered enlarged onion-like structues, in which the internal lamina elastic appears to be megakaryocytes in a bone marrow biopsy material was found to be absent and the external elastic membrane is fragmented (Figure 3) [7]. diagnostic for ET and excluded reactive thrombocytosis (Table 2). The minimum criterion for the diagnosis according to the PVSG in 1975 Erythromelalgic Cerebral and Ocular Ischemic was 1000x109/L [12], which was reduced to 600x109/L in 1986 [13,14]. Manifestations in ET and PV Between 1975 and 1980 we used bone marrow histology as the In the original cohort of 24 patients with myeloproliferative thrombocythemia (11 ET, 13 PVT) aspirin responsive migraine-like S. No Criteria atypical transient ischemic attacks (MIA) and acute coronary syndrome Major criteria A1 Persistent platelet count in excess of 400x109/L. (ACS) were observed in eight patients (5 ET, 3 PVT, Table 1) [3,4]. A Recurrent sudden attacks of unconsciousness lasting for a few minutes A2 Increase and clustering of large megakaryocytes in bone marrow did occur in 3 patients with erythromelalgia (A2, A3, A16). Migraine biopsy like headache followed by recurrent transient hemiparesis lasting for Confirmative criteria half an hour (migraine accompagne) was associated with disorientation B1 Presence of large platelets in a peripheral blood smear B2 Absence of any underlying disease for reactive thrombocytosis and in time [8] in one patient with a history of myocardial infarction. One B case suffered from severe migraine–like headache followed by nausea normal ESR. and emesis after discontinuation of aspirin (A7). On case with a history B3 No or slight splenomegaly on palpation or scan (<15 cm) of myocardial infarction suffered from recurrent transient ischemic B4 Increase of LAP-score and no signs of fever or inflammation attacks (A6). In 1993 we reported on the presenting transient neurologic Exclusion criterion and ocular manifestation in ET patients [9] and subsequently reviewed Ph+ chromosome and any other cytogenetic abnormality in blood or bone marrow cells the literature [10,11]. The transient neurologic and ocular symptoms B. The 1980 RCP major (A) and minor (B) criteria for prefibrotic PV3 in ET are featured by migraine like atypical transient cerebral or ocular A1 Raised red cell mass. Male >36 ml/kg, female >32 ml/kg: PVSG ischemic attacks (MIAs) [9-11]. The main neurologic symptoms in ET 197514 are poorly localized cerebral symptoms of unsteadines, unstable gait, A2 Increased erythrocyte count above 6x1012/L. or attacks of sudden falling without loss of consciousness or weakness, A3 Increase in bone marrow biopsy of clustered, large pleomorphic focal transient cerebral symptoms of mono- or hemiparesis and focal megakaryocytes with hyperlobulated nuclei and increased cellularity transient visual symptoms of monocular blindness [9]. The cerebral or due to increased megakaryopoiesis erythropoiesis or typically trilinear C visual symptoms are accompanied by migraine-like dull or throbbing mega-erythro-granulopoiesis. typical PV bone marrow excludes erythrocytosis618,19. unilateral or bilateral headaches, usually lasting for several hours. B1 Thrombocythemia, persistant increase of platelet >400x109/L None of the patients had a history of migraine. Global visual symptoms B2 Leukocytosis, leucocyte count >109/L and low erythrocyte include scintillating scotomas and blurred vision. All transient sedimentation rate neurlogic and visual attacks had a sudden onset and occurred gradually B3 Raised leukocyte alkaline phosphatase (LAP) score >100, absence rather than all at once and lasted for a few seconds to several minutes. of infection The progression had both a rapid (within seconds) spreading of visual B4 Splenomegaly on palpation or on isotope/ultrasound scanning symptoms and a sequential occurrence of one symptom after another; A1or A2 plus A3 and none of B establishes erythrocythemic PV for instance migraine-like visual symptoms followed by dysarthria, A1 or A2 plus A3 plus one of B establishes PV and excludes erythrocytosis followed by weakness [9]. Progressfion off ftransfienft Themfiparesfis fto sftroke Table 2: The Rotterdam Clinical and pathological (1980 RCP) criteria of essential or permanent blindness did occur if not treated with aspirin. Routine thrombocythemia (ET) defined and used between 1975-1980 [3].

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Number Diagnosis Gender Spleen Spleen Platelets Hb Ht Erythroc BM iron Age yrs scan cm 109/L mmol/l 1012/L stain A1 ET F 60 np 10.6 792 10.4 0.49 6.7 neg A2 ET M 39 np 9.8 887 10 0.51 6 pos A3 ET F 42 - 5 911 8.9 0.47 5.4 pos A4 ET M 47 p 13.5 614 8 0.39 4.5 - A5 ET M 46 np 10.5 939 8.3 0.4 4.4 pos A6 ET M33 p 14.3 742 9.8 0.49 5.8 pos A7 ET M 45 np 11.2 567 9.5 0.46 5.2 pos A8 ET M39 np 10.8 875 8.8 0.43 4.9 pos A9 ET M51 np 11.2 690 8.8 0.45 5.5 pos A10 ET M40 p 14.4 1440 8.6 0.43 4.7 pos A11 ET F 72 p 14.8 1435 9.4 0.46 6.1 neg A12 PTV M 63 np 8 1932 11.1 0.56 6.5 neg A13 PTV F 75 np - 1800 12.1 0.62 7.6 neg A14 PTVb M 61 - 5 952 8.3 0.45 5.6 neg A15 PTVb M 53 p 13.1 636 7.7 0.39 5.4 neg A16 PTV F 60 p 14.5 1065 13.4 0.68 7.9 neg A17 PTV F 49 p 15.8 728 10.9 0.57 7.5 neg A18 PTV M 66 p 15.9 1035 12.2 0.64 7.1 neg A19 PTV M 71 p 15.3 1320 13.3 0.7 6.4 neg A20 PTV M 65 p 14.9 1300 11.9 0.65 7.6 neg A21 PTV F 55 p 14.1 1085 12.1 0.61 7.1 neg A22 PTV F 59 p 12.5 708 11 0.59 7.5 neg A23 PTV F 74 p 14.4 959 13.1 0.72 9.1 neg A24 PTV M 71 p 16.6 609 12.5 0.66 9.9 neg B1 PV/HT M 50 p - 2316 10.2 0.52 6.3 neg B2 PV/HT F 86 p 19 2975 5.3 0.32 4.4 neg B3 ET/HT M 72 np 11.4 1503 6.3 0.31 3.5 pos B4 ET/HT F 45 np 13.8 1074 8.1 0.41 4.6 neg B5 ET/HT M 51 np 10.1 1080 8.3 0.42 4.9 - B6 PV/HT M 61 np 11.4 737 9.9 0.49 5.1 neg

Table 3a: Clinical and peripheral blood data in 14 ET and 16 PTV patients (Source Michiels Thesis 1981[1];: A1/24 complicated by erythromelalgia and B1/6 hemorhagic thrombocythemia (HT) [3]. ET: Essential Thrombocythemia, PTV: Polycythemia Thrombocythemia Vera. b= Treated ith Busulphan, np: Not Palpable, p=Palpable Source. Michiels J.J. Thesis, Erythromelalgia and Thrombocythemia, Rotterdam 198:160-163. pathognomonic clue to PV on top of the 1975 PVSG criteria for the macrovascular complication at normal hematocrits of 0.40 or less. A diagnosis of polycythemia vera [14,15]. Erythrocyte volume (red cell hematocrit of <0.45 is not low enough to prevent major thrombosis. mass RCM) was measured using Cr51 (natriumchromate) labeled The microvascular syndrome associated thrombocythemia persist in autologous erythrocyte1. The RCP modifications in 1980 of the 1975 PV in hematological remission by phlebotomy alone. Platelet mediated PVSG criteria for PV14 include 4 main changes (Table 2) [3,4]. microvascular thrombotic disease in ET and PV is best controlled by First, the major criterion O2-saturation of >92% is deleted since a low dose aspirin (100 mg/day) or selective reduction of platelet counts bone biopsy differentiates between PV and erythrocytosis9. Second; to normal, (<350x10 9 /L). We have elucidated the pathophysiology splenomegaly is replaced by bone marrow histology as a major criterion of aspirin sensitive platelet thrombophilia in ET and PV patients in (A3, Table 2). Third, the RCP diagnostic set used splenomegaly as a several original basic research publications and in one recent insight minor criterion (Table 3a). Fourth, we skipped raised B12 (>900 ng/L) review articles [16-20]. or raised B12 binding capacity (>2200 ng/L) as completely irrelevant In the period of January 1975 to December 31, 1980, we for the diagnosis of early and overt stage PV (Table 2). prospectively studied the laboratory and bone marrow features in 30 Prospective Laboratory Investigations and Bone consecutive early prefibrotic stage MPN patients, who presented with Marrow Features of Thrombocythemia in ET and PV erythromelalgic thrombotic thrombocythemia (ETT), 14 ET and 16 PV patients (Table 3b) [3,4]. The clinical features of erythromelalgia Platelet-mediated arteriolar inflammation and platelet thrombi caused by platelet-mediated arteriolar inflammation and thrombosis (erythromelalgia) did not occur in reactive thrombocytosis, which in thrombocythemia of the ET and PV cases have been reported in strongly indicates not only a quantitative but also a qualitative disorder great detail in the Annals of Internal Medicine, 19854. The mean age of platelet hyperfunction in thrombocythemia [3,4,8,10]. This unknown of 30 ETT patients (ET and PV) was 56.7 (range 33-96) years. Eleven platelet defect in thrombocythemia in ET and PV paients is postulated of 14 ET patients had platelet counts below 1000x109/L, in which the by Michiels and co-workers to lead to an increased risk of microvascular RCP diagnosis of ET (Table 2) would have been overlooked by the thrombotic complication and microvascular and macrovascular PVSG criteria at that time. Spleen size on scan was slightly increased thrombotic complication in thrombocythemia of various MPNs [16- in 5 of 14 ET and in 13 of 16 PV patients. Leukocyte count counts was 20]. Phlebotomy alone in PV significantly reduced the incidence of increased (>10x109/L) in 5 out of 14 patients with ET and in 14 of 16

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Number Leukoc LAF RCM Ery ESR BM BM1 BM1 BM M/E BM iron 109/L score volume megakar cellularity reticuline ratio stain A1 ET 10 183 31 2 1+ N N 2.1 neg A2 ET 9 155 - 0 1+ N N 2.9 pos A3 ET 8 109 - 1 2+ 2+ 1+ 3.3 pos A4 ET 9 101 - 4 - - - - A5 ET 16 128 26 2 1+ N N 3 pos A6 ET 7 139 - 6 1+ N N 3.1 pos A7 ET 8 127 - 3 1+ N N 2.9 pos A8 ET 10 38 - 3 2+ 1+ 1+ pos A9 ET 10 103 - 52 1+ 1+ 1+ 3.2 pos A10 ET 11 60 - 5 1+ N N 4 pos A11 ET 13 113 32 3 2+ 1+ 2+ 1.9 neg A12 PV 10 207 59 75 1+ 2+ 1+ 1.1 neg A13 PV 28 193 - 2 2+ 2+ N 1.7 neg A14 PV 13 236 - 4 2+ 2+ 2+ 1.1 neg A15 PV 11 103 - 63 2+ 1+ 1+ 1.6 neg A16 PV 17 243 45 3 2+ 2+ 1+ 1.2 neg A17 PV 8 186 60 1 1+ 1+ 1+ 0.6 neg A18 PV 14 184 63 2 2+ 1+ 1+ 2.2 neg A19 PV 16 219 50 1 2+ 2+ 1+ 3.3 neg A20 PV 18 128 38 3 2+ 1+ 1+ 1.2 neg A21 PV 13 170 43 1 2+ 2+ 2+ 2.1 neg A22 PV 17 168 42 0 2+ 2+ 2+ 1.3 neg A23 PV 9 219 54 2 2+ 2+ 1+ 0.5 neg A24 PV 18 215 82 0 2+ 2+ 1+ 1.3 neg B1 HT 46 - - 0 2+ 2+ 2+ - neg B2 HT 24 235 38 1 2+ 2+ 2+ 0.6 neg B3 HT 8 49 - 4 2+ N 1+ 2.2 pos B4 HT 7 105 - 7 1+ N N 2.5 neg B5 HT 10 47 - 2 2+ 2+ 2+ - B6 HT 8 121 - 36 1+ N 1+ 1.8 neg

Table 3b: Peripheral blood and bone marrow data in 14 ET and 16 PV patients (Source Michiels Thesis 1981 [1]): A1/24complicated by erythromelalgia, and B1/6 hemorrhagic thrombocythemia (HT) [3]. Source. Michiels J.J. Thesis, Erythromelalgia and Thrombocythemia, Rotterdam 1981:160-163. PV patients. LAP score was increased (>100) in 12 out of 14 ET and in all PV patients. Increase of clustered large megakaryocytes and a normoblastic erythropoiesis (decreased M/E ratio) was present in bone marrow smears and biopsies of 14 ET and 16 PV patients (Table 3b, Figures 6 and 7). The myeloid/erythrocyte (M/E) ratio was shifted towards increased erythropoiesis in half of ET and most PV patients. The mean M/E ratio in 14 patients with early stage ET was 1.95 (range 0.6-4.0) as compared to 1.53 (range 0.6-3.3) in 16 PV patients. A normocellular bone marrow picture with increase of clustered pleomorphic megakaryocytes and no increase of cellularity (Figure 5) was seen in 7 of 14 ET and in one of 16 PV patients. A moderate increase of cellularity (1+=60-80%) in the bone marrow due to increased erythropoiesis (=decreased M/E ratio) leading to a ET/PV picture was seen in 3 ET and 4 PV patients (Figure 7). A typical PV bone marrow picture with pronounced increase of cellularity (2+=80-100%) due to predominantly increased erythropoiesis or trilinear hypercellular hematopoiesis (megakaryo/ erythro/granulocytic: panmyelosis Figure 6) was seen in 2 of 14 ET patients and in 11 of 16 PV. These results indicate that bone marrow histopathology on its own is not reliable to clearly differentiate between ET and PV but appeared to be a powerful tool to differentiate ET Figure 6: Presence of large platelets in peripheral (EDTA) blood smear (left top), increase of clustered enlarged megakaryocytes in a normocelluar ET and PV from all variants of primary or secondary erythrocytosis and bone marrow with stainable iron (left bottum). Local increase of erythropiesis reactive thrombocytosis with a sensitivity and specificity near to 100% in areas of loose clustered pleiomorphic megakaryoctyes is seen in essential [3,4]. The morphology of large pleomorphic megakarocytes were not thrombocythemia (ET: right panels). different in ET and PV except that pleomorphism of megakaryocytes

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Erythromelalgic Thrombotic Thrombocythemia (EET) and Hemorrhagic Thrombocythemia (HT) In the period we reported the erythromelalgic, thrombotic and hemorhagic manifestations in 50 consecutive cases of thrombocythemia in 30 ET and 20 PV patients 10: 16 ET and 15 PV patients suffered from erythromelagic thrombotic thrombocythemia (ETT). Besides erythromelalgia 16 thrombocythemia patients suffered from additional transient arterial ischemic or thrombotic complications involving thebrain or heart : transient cerebral neurologic ischemic attacks (TIA) in 3, and migraine-like atypical TIAs in 8 ; myocardial infarction in 2; amourosis fugax in 2 and venous thrombosis was not observed. Hemorrhagic thrombocythemia (HT) was diagnosed in 8 patients without erythromelalgia. The presenting features of HT were bruises and ecchymoses in 4, recurrent epistaxis in 2 bleeding after tooth extraction in 2 and gatsrointestinal bleeding in 2. Paradoxical occorrence of erythromelalgia and bleeding symptoms (ETTT plus HT) was observed in 5 thrombocythemia patients. The laboratory data at time of presentation in 30 ET and 20 PV patients are shown in Figure

8. Hemoglobin levels were normal in ET and elevated in PV. Leukocyte Figure 7: ET (left upper), PV (right upper, left bottum), and trilineear PV (right counts were normal or increased in ET and PV. The leukocyte alkaline bottum) bone marrow histology seen in ET and PV patients. Pleiomorphic phosphatase score was much more elevated in PV than in ET (Figure megakaryocytes in ET (upper panels) have similar hyperlobulated nuclei as in 8). PV (bottum panels). Dense clustered pleiomorphic megakaryocytes (PV/RF 2, right bottum) is seen in advanced PV show dysmorphic nuclei but not cloud- Seventeen of 31 patients with erythromelalgic thrombotic like. Please note relative increased erythropiesis in ET (upper panels). thrombocythemia had platelet counts below 1000x109/L with the lowest count of 420x109/l (Figure 8) [10]. Platelet counts in 8 HT patients and 5 ETT/HT patients with paradoxal erythromelalgia and bleeding 9 Hb Leucocytes LAF Platelets Uric acid had platelet counts between 850 to 3000x10 /L and were in excess of 9 mmoI / I x 10 / I scores x 10 / I mm0I / I 1000x109/L in 12 of 13 thrombocythemia patients (Figure 9). Budde PT PV PT PV PT PV PT PV PT PV 250 et al discovered that increased platelet count far above 1000x109/L in HT was associated with an acquired von Willebrand syndrome type 2A [21,22]. Michiels and co-workers prospectively documented in ET and PV patients the details of paradoxical occurrences of erythromelalgic 20 20 200 2000 microvascular thrombotic manifestation and spontaneous 0. 70 mucocutaneous bleeding at platelet count above 1000x109/L during

0. 60 long-term follow-up caused by an acquired von Willebrand syndrome 15 15 1500 type 2A, which disappeared after correction of platelet count to normal 0. 50 (Figures 10-12) [20,23-27]. 0. 40 Platelet Lowering Agents in ET: Low Dose Busulphan to 1000 10 10 100 0. 30 Control Platelet Number and ETT Symptoms 0. 20 The European Organization on Research and Treatment of Cancer (EORTC) conducted phase III study in previously untreated PV 500 5 5 50

Number of patients 12 asymptomatic 10 Figure 8: Laboratory findings at time of presentation in symptomatic with major hemorrhages thrombocythemia subdivided in 30 PT (=ET) and 20 PV patients seen between 8 1975 and 1985. Hb levels are normal in PT and elevated in PV 16. Leukocytes paradoxal erythromelalgia and hemorrhages were normal or elevated in PT and PV. LAP score was much more elevated in 6 PV than in PT with normal values in 6 PT patients. Platelet counts were were with erythromelalgia between 400 and 1000×10/L in the majority of the 30 PT patients. Serum uric 4 acid levels werenormal in PT and usually elevated in PV. 2

0 was more pronounced in the hypercellular (80-100%) bone marrows 0 500 1000 1500 2000 2500 3000 x109 /1 of PV and ET patients with advanced myeloproliferative disease Platelets (Figure 7) [3,4]. We could conclude in 19813 that RCP defined ET and Figure 9: Frequency distribution of platelet counts in 50 patients (30 ET and 20 PV patients show overlapping bone marrow pictures with increase PV) ssociated with erythromelalgic thrombotic thrombocythemia (ETT, N=31), hemorrhagic thrombocythemia (HT, N=8) and paradoxical occurrence of ETT of pleomorphic clustered megakaryocytes and varying degrees of and HT (N=5) Michiels et al. increased erythropoisesis at time of diagnosis (Figures 6 and 7).

Page 8 of 16

in 20, foot/leg ulcers in 4, atypical transient neurologic or ocular BEFORE BUSULFAN TREATMANT ischemic manifestations in 11, major thrombosis in 8. Hemorrhagic 1500 9 x 10 /1 thrombocythemia (HT) had occured in 8 at platelet counts in excess platelets of 1000x109/L. Reduction of platelet count by low dose busulphan to less than 400x109/L and no aspirin resolved all vascular occlusive Relapse TOTAL DOSE BUSULFAN still in disease and the disappearance of bleeding [30]. Cox regression analysis (mg) Remission 500 100 revealed two prognistically important features in ET: age had a strong 1000 90 inverse correlation with survival and vascular occlusive symptoms

400 80 REMISSION correlated with a better survival. The number of death was 2.1 times DURATION MONTHS AT RELAPSE 70 than of a comparable control group and death from thrombosis was (1985) ( 1985 ) DURATION 300 60 not significantly different from the number expected [30]. The natural BUSULFAN TREATMENT 50 history was featured by progression of ET into PV occurred in 9% 500 DAYS

AFTER BUSULFAN 200 40 and to myelofibrosis in 24% with death from myelofibrosis markedly TREATMENT increased [30]. 30

100 20 From 1974 to 1986 the Rotterdam MPN Working Group treated

10 20 symptomatic ET patients with aspirin and one course of busulaphan

0 0 0 The symptomatic microvascular thrombotic circulation disturbances

Figure 10: Clinical case 1 Long-term clinical observation of microvascular ranged from erythromelalgia (N=18) , atypical and typical transient circulation disturbances *E, AF, TIA and MI) in a 62 year old female with ET ischemic attacks (N=6), and acute coronary syndromes (N=3) (Hb 10.3, Ht 0.50, leukocytes 10×109/L, LAF-score 100, platelets varying from were [31,32]. Mean platelet count before busulphan treatment was 9 687-767×10 /L) related to platelet counts and the effect of different treatment 9 strategies in a 15 year prospective follow-up of a typical case of essential 1009x10 /L,( range 545 to >1525, Figure 13). One course of busulfan thrombocythemia (ET). Erythromelalgia (E) amaurosis fugax (AF, transient treatment was started at a dose of 4 to 6 mg/day at platelet counts ischemic attacks (TIA) and recurrent myocardial infarctions (MI;s) all caused >1000x109/L (Figure 10). At platelet count just below 1000x109/L by platelet-mediated thrombotic processes in the end-arterial microcirculation busulfan dose was reduced to 4 or 2 mg/day under carful blood cell did occurr during two periods at a platelet count around 400×109/L. Aspirin one dose daily relieved all erythromelalgic thrombotic microvascular ischemic circulation disturbances including E, AF, TIA and MI, which was accompanied with a significant rise of platelet count of about 200×109/L, from around 400 Platelets 9 AF TIA MI’S to around 600×109/L at two occasions. After reduction of platelet count to x 10 / L completely normal values (<300×109/L)with busulfan and discontinuation E of aspirin according to our protocol, the patient remained asymptomatic for several years until microvascular circulation disturbances did recur at platelet 800 counts around 400×109/L. The patient ultimately died of post-myocardial heart failure in 1991. 700

600 patients *n=293). Previously untreated PV patients were randomized for P32 and busulfan in the years 1967-1978 [28]. The hematologic 500 objective response criteria were given in terms of hematocrit values between 0.42 and 0.47. With a median follow-up of 9 years, busulfan 400 treatment (mean dose 310 mg) was better than P32 (mean dose 5.9 300 mCi) [28]. There were fewer thrombotic episodes with busulfan than with P32 due to better control of the thrombocythemia with busulfan: 200 25 of 149 treated with P32 died of vascular complication compared to 8 of 145 treated with busulfan, but the incidence of acute leukemia 100 after a median follow-up of 9 years was equally low and not statistically different between the two agents (2/140 P32 versus 3/145 busulphan 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 [28]. Overall survival at 10 years was significantly better with busulphan E= Erythomelalgia + =present - =absent (74%) than with P32 (55%). This difference in overall survival was AF = Amourosis Fugax TIA = Transient Ischemic Attack MI ‘S = Myocardial Infarctions = Aspirin, = Busulfan and = Coumarin treatment due to a much higher frequency of vascular accidents in the P32 arm because of a poorer control of hemoglobin values and platelet counts. Figure 11: A 72 year-old woman with a three years history of burning painful red or blue toes and forefoot sole of the right foot (erythromelalgia) presented in Consequently, twenty five of 149 patients treated by P32 died from Decembrer 1978 with spontaneous hemorrhages of large ecchymoses on the vascular complications not on aspirin compared to 8 of 145 PV patients chest and subcutaneous hematotomas. Paradoxical occurrences of platelet- treated by busulphan not on aspirin [28]. mediated thrombosis and bleeding in this case with thrombocythemia (ET with features of PV in the bone marrow: prodromal PV) 1978 peak 1 in the figure: The London Myeloproliferative Study Group of Wetherley-Meinet first episode of erythromelalgia for 3 years followed by simultaneous occurrence al used the alkylating agent buslphan more judiciously as an effective of thrombotic thrombocythemia and hemorrhagic thrombocythemia at platelet counts around 1500×109/L in 1982 and 1987. During periods of busulphan and safe platelet reductive agent for the control of thrombocythemia induced remission of thrombocthemia (platelet counts below 400×10/L) the in PV [29], and for the control of platelet cout in symptomatic cases patients was asymptomatic when not on aspirin. Peak 2 and 3 in the figure: of ET30. In the London ET study, Wetherley-Mein and co-workers recurrence of second and third episode of thrombotic thrombocythemia at platelet counts between 600 and 800×109/L. 1992 peak 4 with a fourth reported on the effective treatment with busulphan in 37 cases (mean episode of thrombotic and hemorrhagic thrombocythemia at platelet counts of age 60.5, range 30-89 years) with PVSG defined ET with platelet counts 1040×109/L and aspirin-induced increase to around 1500×109/L associated with between 615-2450x109/L [30]. The vascular occlusive symptoms at acquired von Willebrand factor deficiency type 2A (acquired von Willebrand presentation in 19 patients were painful feet and toes (erythromelalgia) syndrome: AVWS (figure 12).

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The Spectrum of ETT and HT in ET and PV patients:

9 Therapeutic Implications Platelets x10 /1 Busulfan and Aspirin Aspirin In 1980 [33-35] Michiels reviewed 100 case histories of primary Hydroxyurea 1500 hemorrhagic thrombocythemia (HT) and 99 cases of erythromelalgic thrombotic thrombocythemia (ETT, 67ET and 32 PV). The bleeding manifestations in 100 HT cases ranged from gastrointestinal chronic 1987. Second relapse of thrombocythemia. occult blood loss, melena and hematemesis to mucocutaneous bruises, Platelets 664x109 /L. Sudden attacks of unsteadiness. Dysarthria and amourosis fugax. hematomas, ecchymoses, gum bleedings and secondary bleeding 1982. The cerebral ischemic attacks did not recur during aspirin. [33-35]. HT was usually associated with significant leukocytosis and 1000 First relapse of thrombocythemia. Platelets 584x109 /L. splenomegaly and the platelet count at time of bleeding in 100 HT cases Recurrent transient monoparesis in ranged from 800 to above 4000x109/L. The manifestations in 99 ETT (67 the right arm. Facial palsy and blurred vision. ET and 32 PV) patients included erythromelalgia, acrocyanosis, digital gangrene, amaurosis fugax, transient ischemic attacks, stroke, angina pectoris and myocardial infarction, superficial thrombophlebitis and 500 deep vein thrombosis. The platelet count at time of ETT in ET and PV patients ranged from 400 to 2000x109/L in ET patients and from 350 to 1 ETT=Erythromelalgic Thrombotic Thrombocythemia. HT=Hemorrhagic Thrombocythemia; ET: Essential Thrombocythemia; 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 year PV=Polycythemia Vera. Figure 12: Left. Acquired von Willebrand syndrome at high platelet count during VWF:RCo=Von Willebrand Ristocetine Cofactor Activity; VWF: period 4 in Figure 11. Period 4 was followed by hydroxyurea induced reduction of platelet count to normal which was associated with the disappearance of CB=Von Willebrand Factor Collagen Binding. thrombohemorrhagic manifestations and the disappearance of the AVWS. Right. The VWF: CB deficiency is best correlated the degree of loss of large The final follow-up of the Rotterdam MPD Working Group VWF multimers at increasing platelet count in a low SDS resolution gelwith (1975-1995) on the efficacy and safety for the prevention and paradoxical occurrence of thrombohemorrhagic thrombocythemia in period 4 treatment of thrombotic complications in 68 ET patients seen in the of Figure 11. Academic Hospital Rotterdam has been critically evaluated by Perry van Genderen et al (1997) [36,37] independent from the principal counts every week. Busulphan was discontinued in all 20 ET cases when investigator (Michiels). In this prospective observational Rotterdam platelet counts reached the upper limit of normal (350x10/L (Figure 13).

At time of complete remission of ET, aspirin was discontinued to test 1.0 U/Ml VWF=RCo the hypothesis whether control number to normal was associated with VWF=CB cure and no recurrence of erythromelalgic circulation disturbances. As 0.6 shown in Figure 9 the mean platelet count before and after busulfan treatment was 1009x10/9L (range 545-1525) and 241 (range 159-315) x109/L respectively. At time of normal platelet count (<350x109/L)

500 1000 1500x 109 /I Platelets erythromelalgia did not recur after discontinuation of aspirin. The 9 NP P:1400 1250 890 575x10 /L P:295 NP total dose burden of only one course of busulfan treatment ranged Figure 13: Platelet counts before and after one course of busulphan treatment from 106-484 (mean 315) mg. The durarion of the busulfan course in 20 cases of essential thrombocythemia (ET) and at time of relapse of ET. ranged from 44 to 218 (mean 116) days. All 20 patients with busulphan Total dose and treatment duration of busulphan courses and remission duration after discontinuation of busulphan in ET patients [31,32]. 250x109/L in PV induced remission of ET remained asymptomatic after discontinuation patients (Figure 14) [33-35]. of both busulfan and aspirin as long as platelet counts were normal for a follow-up period from 10 to 60 months (Figure 10). At time of relapse of ET (platelets >400x109/L) in 12; 8 became symptomatic (mainly Thrombotic complications Bleeding complications Events Events Treatment Strategy Person/yrs person-years person-yrs erythromelalgia and atypical TIA in a few) at slight increased platelet (n) (n) 9 count of 410, 450, 490, 500, 515, 545, 548 and 635x10 /L indicating Asymptomatic 14 Patients: the need of low dose aspirin (100 mg), and 4 remained asymptomatic Watchful waiting 127 27* 33.3 2 1.6 at platelet counts of 577, 600, 648 and 725x109/L (Figure 10). Based Symptomatic 54 patients: on these prospective intervention studies, we decided in 1986 to treat Low-dose aspirin 139 5 3.6 10*** 7.2 our ET patients with aspirin at time of presentation, during follow- Platelet reduction 113 10** 8.9 2 1.8 up, and at relapse as long as platelet counts were between 400 and Low-dose aspirin + 40 0 - 4 10 1000+200x109/L. In 1986, hydroxyurea at that time became the drug platelet reduction Total 419 42 18 of choice for clear indications to reduce platelet count from above to below 1000x109/L with continuation of low dose aspirin (Tables 4 and Table 4: Incidence of thrombotic and bleeding complications in the prospective 1975-1996 Rotterdam study of 68 ET patients during a median follow-up of 6.7 5). At that time bleeding symptoms, aspirin side effects and platelet years according to treatment strategy (Van Genderen et al 1997) [36,37]. Mean counts in excess of 1000 to 1500x109/L were clear indications for platelet count *=610, range 410-831×109/l at time of thrombotic event. **=624 ± 9 *** 9 correction of platelet counts in ET and PV patients [10,31,32]. 255×10 /l at time of thrombotic event. =1737, range 661-3460×10 /l at time of bleeding event (Table 5).

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Platelets Platelets Platelets Platelets 400-1500x10 9/L 400-1000x109/L 400-1000x109/L >1500x109/L Low risk ETT/HT Standard risk ETT High risk ETT/HT High ETT/HT risk JAK2 wild type JAK2V617F mutated JAK2V617F mutated All variants ET Asymptomatic Major thrombosis >1000 x10 9/L and minor thrombosis/ Microvascular Microvascular disturbances only* Spontaneous bleeding or elicited by bleeding=high dsturbances aspirin No vascular risk No vascular risk Vascular risk(s) No vascular risk No bleeding risk No bleeding risk Bleeding risk All ages* All ages* Age >65 years* All ages Aspirin uncertain Low dose aspirin Platelet reduction to normal or near Platelet reduction to <1000x109/l Wait and see? 50 to 100 mg/day normal Intermediate risk Aspirin primary prevention? Microvascular disturbances and platelet ET patients and count between 1,000 and 1,500 x109/l When platelets <1,000 x 109/l continue their physician Continue aspirin* with clear indication aspirin*, side or add aspirin usually prefer the use of low dose effects platelet reduction aspirin *At platelet counts in excess of 1,000+250x109/l aspirin will usually elicit bleeding symptoms, which disappear after reduction of platelet counts to below 1,000 x 109/l with continuation of aspirin (Figure 4). If side effects of aspirin (gastritis, GI-bleeding occur) reduce platelet to normal and consider indomethacin 25 mg TID. Table 5: Low, intermediate and high thrombohemorrhagic risk stratification of ET, ET with features of early PV in blood and bone marrow (prodromal PV) and classical PV patients: a flexible approach towards therapeutic implications with reference to platelet counts [11].* At platelet counts in excess of 1,000+250x109/l aspirin will usually elicit bleeding symptoms, which disappear after reduction of platelet counts to below 1,000 x 109/l with continuation of aspirin (Figure 4). If side effects of aspirin (gastritis, GI- bleeding occur) reduce platelet to normal and consider indomethacin 25 mg TID.

Activity (U/ml ) 2.0

VWF:RCo VWF:CB

1.0

0.6

N 0 0 1000 2000 3000 4000 1 .50 Platelets x 109 /I

PLATELET ETT ETT + HT COUNTS 1 .00 x 109 /L : 0 350 500 1000 1500 2000 3000 4000 400 Ratio 0 .50 HT ASPIRIN

0 .00 0 1000 2000 3000 4000 ETT: ERYTHROMELALGIC THROMBOTIC THROMBOCYTHEMIA 9 HT: HEMORRHAGIC THROMBOCYTHEMIA Platelet count (x 10 /I)

Figure 14: left The Cigar of ETT and Wedge concept of erythromelalgic thrombotic thrombocythemia (ETT) and hemorrhagic thrombocythemia (HT) [10,34,35]. The transition of ETT into HT is related to the increase of platelet counts from below to far above 1000×109/L caused by a platelet mediated proteolysis of large von Willebrand factor (VWF) mutimers resulting in a case with post-spenectomy HT (described in detail by van Genderen et al [23]) was associated with an acquired von Willebrand disease (AVWD) type 2A.b: Right Loss of large VWF multimers at increasing platelet count in a case of post-splenectomy HT (described in detail by Van Genderen et al [23]) was associated with low VWF:RCO/VWF: Ag (open circles) and VWF:CB/VWF: Ag (black dots) ratios indicative for a typical acqiured von Willebrand disease (AVWD) type 2 A [20,23,24].Reduction of the platelet counts to normal with hydroxyurea resulted in disappearance of bleeding symptoms and correction of the functional VWG:RCo and VWF:CB to normal, normalization of the bleeding time, and reappearance of all large VWF multimers [23]

ET study the 68 consecutive ET patients were followed between 1975 event were between 410 and 831, mean 610x109/L. Only four of the to 1995 for a total of 419 patient years (Table 4) [36,37]. At time of initially 14 asymptomatic ET patients remained asymptomatic after at presentation 54 patients had ET related microvascular thrombotic least 30 person-years follow-up. In the treatment group of low-dose complications at platelet counts between 575 and 1031, mean 750x109/L aspirin alone, 5 thrombotic events during a follow-up of 179 patient and treated with low-dose aspirin, a platelet lowering agent (busulphan years, and 10 bleeding episodes occurred in ET patients receiving low or hydroxyurea) or both. The 14 ET asymptomatic ET patients suffered dose aspirin monotherapy during a follow-up of 139 patients years at from 27 thrombotic and two bleeding episodes occurred during a platelet counts between 661 and 3460 (mean 1737)x109/l (Table 4). The follow-up of 127 patient years; platelet counts at time of thrombotic main conclusion from this observation is that aspirin alone in ET at

Page 11 of 16 platelet counts above 1000x109/l in general daily practices is not safe The characteristics of the thrombotic events in the retrospective enough in terms of bleedings elicited by aspirin indicating the need to Bergamo cohort of 100 patients were the following: add one of the non-leukemogenic platelet lowering agents pegylated The age distribution of this cohort of ET did not reflect real life interferon or anagrelide [36,37]. In patients receiving platelet lowering experience since the number of young ET patients was artificially agents either busulfan or hydroxyurea and no aspirin, 10 thrombotic manipulated to one third in the young age group to reach statistical complications occurred during follow-up of 40 patient years at significance. The risk for thrombotic complication was artificially low platelet counts above 400x109/L (624 ± 25x109/L, Table 4), indicating (1.7%) in thrombocythemia at young age below 40 years, but was high that inadequate control of platelet number in thrombocythemia of at age of >60 years (15%) not on aspirin, and moderately increased ET and PV patients is associated with a persistent high incidence of (6.3%) in the age group of 40 to 60 years, which is not significantly microvascular disturbances including MIAs when not on aspirin at different from the thrombotic risk of 15% in the age group of >60 years plateletcounts above 350 to 400x109/L (Figure 14). In this landmark [40]. Please note that in the Dutch prospective ET and PV studies study of Van Genderen et al, age above 65 years was not a risk factor for the risk of thrombosis is untreated ET and PV is not age dependent thrombotic recurrences in ET when on low dose aspirin [36,37]. In the and causally related to platelet-mediated thrombosis in the various 1986 London study of 37 ET cases treated with low dose busulphan for stages of prefibrotic and early fibrotic MPN disease of ET and PV the treatment of ETT and HT not on aspirin (mean age 60.5, range 30- 9 patients [36,37,40]. The type and number of 25 arterial and 3 venous 89 years ,platelet between 615-2450x10 /L, Figure 14) [25] all vascular thrombotic episodes in 20 out of a historical cohort of 100 untreated occlusive disease and bleeding resolved at time of platelet correction ET patients in the 1990 Bergamo study were mainly microcirculatory to normal [30]. Age had a strong inverse correlation with survival events including digital ischemia, transient ischemic attacks, superficial but vascular occlusive symptoms correlated with a better survival; thrombophlebitis, ususual site of DVT, no stroke, and major progression of ET into PV occurred in 9% and to myelofibrosis in 24% thrombosis only in 4, myocardial infarction in 3 and femoral DVT with death from myelofibrosis markedly increased [30]. in 1 (Table 6) [40]. It has been demonstrated at that time that such The therapeutic implications from the prospective Rotterdam ET microcirculatory disturbances, superficial thrombophlebitis and TIAs studies are completely in line with the concept that microcirculatory are highly sensitive to low dose aspirin but not to coumarin [2]. If left thrombotic complications in thrombocythemia already occur at platelet untreated symptomatic ET patients with microcirculatory disturbances counts in excess of 400x109/L, which are relieved by reduction of platelet including erythromelalgia and atypical TIAs or visual symptoms are counts to normal (<400x109/L) or by control of platelet function with supposed to be at very high risk for digital ischemia, TIAs, stroke or low dose aspirin (Figures 14 and 15). Thrombocythemia with platelet acute coronary ischemic syndromes [1-7]. We concluded in 1995 [36] counts in excess of 1000+250x109/L are frequently associated with the and 1997 [37] that the stratification in low, intermediate and high paradoxical occurrence of thrombosis and bleeding. Mucocutaneous thrombotic risk in the 1990 retrospective Bergamo study40 has been bleedings spontaneously occur at platelet count in excess of 1000 derived from ET patients not on aspirin. The microvascular and major +250x109/L due to an acquired type II-like von Willebrand syndrome thrombotic complications in MPN-T disease of ET and PV patients (AVWS, absence of high and intermediate von Willebrand factor at time of presentation and during follow-up while not on aspirin is independent of the degree of thrombocytosis or MPN disease burden (VWF) multimers) increasing in severity at increasing platelet in terms of splenomegaly, leukocytosis and constitutional symptoms. counts to high levels above 1000 to 1500x109/L (Figures 14 and 15). This means that the stratification in low, intermediate and high Reduction of platelet count by anagrelide, interferon (or hydroxyurea if thrombotic risk in the retrospective Bergamo ET study is based on anagrelide and interferon fail) to less than 1000x109/L will result in the statistic misinterpretation and mystification leading to authorative persistence of microvascular thrombotic events and the disappearance overtreatment recommendation with hydroxyurea for MPN disease of the bleeding symptoms and AVWS (Figures 14 and 15). Correction of the platelet counts to normal is associated with no recurrences of number of Events Events % Age Patient/years microvascularevents and complete correction of the VWF-multimeric patients number pt/yrs pattern and correction of all VWF-parameters to complete normal <40 year 34 118 2 1.70% values. The study of Michiels et al. [3-11,31-34] and Van Genderen 40-60 years 37 112 7 6.30% et al. [23-27,35-57] served as the scientific rationale for the ECLAP >60 years 29 73 11 15% (European Collaboration on Low-dose Aspirin in PV) study design Total events in 20 of 100 ET patients=20% (1997) [33]. The ECLAP study nicely confirmed our concept of the Number of effectiveness and safety of low dose aspirin in ET and PV in particular Cortelazzo et al. 1990 Number of events patients [38,39]. One low dose aspirin reduces the risk of microvasular ischemic Total 20 32 or thrombotic complications from above 50% to less than 5% during Arterial 17 25 6.2 years of follow-up, and thereby prevented the progression from low digital ischemia 7 thrombotic risk to high risk thrombotic in ET and PV [38,39]. Micro: transient ischemic attacks 15 Low, Intermediate and High Thrombotic Risk in ET and Major: stroke 0 PV Not on Aspirin myocardial infarction 3 superficial 3 7 Venous The risk stratification for thrombosis as low, intermediate and high thrombophlebitis 3 thrombotic risk by Cortelazzo et al. in 199040 has been derived from femoral DVT 1 a historical cohort of 100 ET patients not treated with aspirin thereby Unusual localization DVT 3 overlooking the original and updated key references of Michiels et al. Bleeding 4 [3,4,11,31-38,41] on the demonstration of aspirin responsive platelet- complications mediated arteriolar and arterial thrombotic tendency inherent to Table 6: The type and number of microvascular thrombotic events in the 1990 thrombocythemia in ET and PV patients. Bergamo study are very characteristic for untreated thrombocythemia [3].

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POLYCYTHEMIA VERA THROMBOCYTHEMIA VERA

CORONARY, CEREBRAL AND PERIPHERAL VASCULAR DISEASE ASPECIFIC INFLAMMATION AND CONGESTION MAJOR ARTERIAL OCCLUSIVE DISEASE PROSTAGLANDINS OCCLUSIVE ACTIVATION OF PLATELET / FIBRIN COAGULATION THROMBI IN ARTERIOLES

RED ACTIVATED ACTIVATED CELLS PLATELETS GRANULOCYTES HEMATOCRIT SECRETION LAF-SCORE AGGREGATION ACQUIRED VON WILLEBRAND DISEASE

BLOOD PLATELET DERIVED VISCOSITY GROWTH FACTOR

SMOOTH MUSCLE CELL PROLIFERATION FIBROMUSCLAR INTIMAL PROLIFERATION OF ARTERIOLES

PATHOPHYSIOLOGY OF ARTERIOLAR AND ARTERIAL THROMBBOSIS AND INFLAMMATION ISH BASEL 1991 AS MULTICELLULAR PROCESSES IN THROMBOCYTHEMIA AND POLYCYTHYMIA VERA MICHIELS / VAN VLIET

Figure 15: Concept of erythromelalgic thrombotic thrombocythemia (ETT) and hemorrhagic thrombocythemia (HT) in patients with essential thrombocythemia (ET) and polycythemiavera (PV). The broad spectrum of erythromelalgic thrombotic thrombocyhemia in essential thrombocythemia (ET) and polycythemia vera (PV), which reflects a novel distinct disease entity of aspirin-responsive arterial thrombophilia in thrombocythemia of various myeloproliferative neoplasms (MPN) [41]. The platelet-mediated microvascular thrombophilia aggravates into major arterial thromboic events in PV patients when not on aspirin during follow-up. Correction of the hematocrit to around or below 0.40 is associated with significant reduction of major tromboses in PV but with the persistence of the microvascular syndrome of associated thrombocythemia, which can best be prevented and treated by low dose aspirin but not by warfarin. At rising platelet counts to above 1000×109/L thrombocythemia of various MPN is complicated by spontaneous mucocutaneous hemorrhages caused by acquired von Willebrand disease type 2A (Figure 14) [22,23]. in ET and PV patients 42. The so-called high thrombotic risk ET as mainly minor thrombotic complications including microcirculatory defined by a history or presentation of thrombosis at time of diagnosis disturbances in 12, and major thrombosis in 2 similar as in the HU or by reaching the age 60 years is not in line with the real incidence of arm (MI 1, and deep vein thrombosis in 1) [43]. Moreover, 10 of thrombotic risk of regularly treated according to good clinical practice the 14 symptomatic patients in the placebo arm manifested mainly in the Rotterdam ET and PV studies at that time and beyond up to 2013 microvascular disturbances and were not on treatment with aspirin [11]. In our analysis and experiences the high thrombotic risk and its [43]. A direct comparison of HU reaching normal platelet counts vs. persistance in the 2012 International Prognostic Score for ET (IPSET) low dose aspirin alone in ET patients at platelet counts below or around [42] with the indication to treat ET disease with hydroxyurea when 1000×109/L is predicted to be equally effective for the prevention of reaching the age of 60 years alone irrespective of the degree of MPN microvascular circulation disturbances in ET (Figure 14 and 15). In the disease burden is not appicable anymore as targeted 2014 guideline for 1995 Bergamo study, the use of aspirin in the placebo arms as compared ET and PV patients on aspirin with a low or intermediate MPN disease to the HU arm was not associated with increased bleeding events at burden. Clinicians should realise that high thrombotic risk in the 2012 platelet counts around 1000×109/L [35]. The recommendation from IPSET guidelines result in overtreatment of ET and PV patients who this unbalanced RTC (HU vs placebo) to use hydroxyurea in so-called have low or intermediate MPN disease burden. high thrombotic risk ET as first line treatment for ET is not justified and potentially leukemogenic and has led to significant world-wide The 1995 Bergamo study is a prospective randomized clinical trial over-treatment of ET patient who usually have low to intermediate (RCT) of 114 ET patients comparing hydroxyurea vs placebo in so-called MPN disease burden and a normal life expectance [43]. high thrombotic risk ET patient [43]. This RCT is unbalanced since only 69% of the placebo group and 70% of the HU-treated ET patients The combined low dose aspirin platelet reduction strategy to lower received antiplatelet drugs, aspirin (effective in thrombocythemia [3,4] platelet count to about values around 600x1012/L (near normal platelet or ticlopedine (not effective in thrombocythemia [3,4]. The results concept) in symptomatic ET-patients by anagrelide or, interferon show that 2 of 56 high thrombotic risk ET patients on hydroxyurea had (hydroxyurea when anagrelide and interferon failed) is most effective major thrombotic events (one stroke, one myocardial infarction) and in daily practices for the treatment of thrombocythemia in ET and that 14 of 58 high thrombotic risk ET patients in the placebo group had PV [11]. Prospective studies comparing this concept of near normal

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Patients PV N=323 ET N=639 occuring at diagnosis or follow-up when not on aspirin48. According JAK2V617 mutation status hetero homozygous hetero wild type to these exclusion and inclusion criteria, the clinical profile and the V617F Number of patients 219 104 368 257 incidence of major thrombotic events in 188 JAK2 homozygous V617F At diagnosis MPN patients (JAK2 mutation above 50% in 104 PV and 14 ET) V617F Major arterial events 21% 15,4% 21.70% 10.50% and in 587 heterozygous (JAK2 mutation less than 50% in 219 PV Venous events 6.40% 2.90% 7.90% 4.70% and 257 ET) and 257 wild type ET patients was assessed and calculated in Table 7 and Figure 16. Homozygous JAK2V617F positive patients with During 10 years follow-up (not on aspirin) an allele mutation load above 50% were older, had higher leukocyte Major arterial events 10.10% 12.50% 6.30% 5.80% counts, hematocrits and larger spleen volumes indicating advanced Venous events 4.10% 7.70% 6.30% 2.70% MPN disease [48]. One hundred seventy-six patients (18.3%) had a Total during life time follow-up major thrombotic event at diagnosis with a similar frequency in PV Major arterial 31.10% 27.90% 28% 16.30% (19.2%) and ET (17.8%). A similar incidence was found in our analysis Venous 10.50% 10.60% 14.20% 7.40% of the literature in 1241 ET patients not on aspirin from 14 retrospective Table 7: Major cardiovascular events at diagnosis or during long-term follow-up studies 50. During long-term follow-up, major thrombosis (usually in 323 PV and 639 ET patients according to the JAK2V617F mutation status in not on aspirin) occurred in 122 patients (12.7%), corresponding to the retrospective study of Vannucchi [48]. Only major thrombotic events were retrospectively recorded not including the erythromelalgic peripheral and migraine 14.9% in PV and 11.6% in ET patients and hemorrhages at diagnosis like cerebral ischemic events [48]. In 14 homozygous ET patients total major manifested in 55 (5.7%) patients, 5.3% in PV and 6.0% in ET20. The arterial and venous events occurred in 78.6% and 57.1% respectively. overall incidence of major thrombotic events during 10 years follow- up usually not on aspirin was about 20% in ET heterozygous for the platelet count strategy versus low dose aspirin alone is warranted. The JAK2V617F mutation and in about 10% for JAK2 wild type ET [48]. prediction is that the near normal platelet count strategy in symptomatic A hemorrhage during follow-up was recorded in 45 (4.7%) ET/PV JAK2 mutated ET, and ET mimicking PV (prodromal PV) seems to be patients. A similar incidence of hemorrhages was found in our analysis far superior to low dose aspirin alone, simple because low dose aspirin will significantly increase the risk of bleeding complications at platelet 9 counts around and above 1000 x 10 /L (Figures 14 and 15). Polycythemia Vera Thrombosis and Bleeding Risk in JAK2 Mutated ET and 1.00 PV and JAK2 Wild Type ET 0.95 0.90 With the advent of molecular screening of MPN patients it should 0.85 be realized that WHO-ET patients with a JAK2V617F mutation load Homo 0.80 of less than 50% of the granulocytes are indeed heterozygous for the Probability Hetero JAK2 mutationl [44-46]. However, WHO-PV patients with a JAK2V617F 0.75 mutation load of less than 50% of the granulocytes proved to be combined 0.70 heterozygous homozygous positive for the JAK2V617F mutation [44-46]. 0 360 720 1080 1440 1800 2160 2520 2880 3240 3600 Days Vannucchi et al determined the JAK2V617F mutation allele burden as Pts at risk expressed in percentages of JAK2V6717F mutated granulocytes of 173 PV 219 183 143 116 99 78 61 54 42 38 31 Hetero patients at diagnosis47. The JAK2V617F allele burden in granulocytes 104 91 84 77 70 59 54 44 36 32 26 Homo ranged from 1-25% in 33%, from 25-50% in 29%, from 50 to 75% in 20% and from 75 to 10% in 18% and treatment consisted of phlebotomy in 49% and cytoreductive therapy (mainly hydroxyurea) in 51% [47]. HR (95%CI) P Essential Thrombocythemia The JAK2V617F allele mutation burden correlated with MPN disease WT 1.0 ----- Hetero 1.49 (0.88-2.52) .13 activity in terms of stimulated erythropoiesis by higher hematocrit and 1.00 Homo 3.97 (1.34-11.7) .013 erythrocytes, lower MCV, serum EPO and ferritine, and stimulated 0.95 myelopiesis by higher leukocytes, serum LDH and LAP score [47]. 0.90 V617F Comparing by statistics alone the PV patients with low JAK2 (1 to 0.85 WT V617 50%) versus high JAK2 (50-100%) allele burden, the relative risks 0.80 Probability Hetero (RR) for MPN disease burden increased from 1 to 4 for pruritis, from 0.75 1 to 4 for palpable splenomegaly and from 1 to 4 for spleen sizes above Homo 0.70 15 cm length diameter on scan [47]. 0 360 720 1080 1440 1800 2160 2520 2880 3240 3600 Days In a subsequent retrospective study Vannucchi et al assessed in Pts at risk 2007 the incidence of thrombosis related to the JAK2 allele burden in 257 228 202 186 159 135 116 92 84 78 66 WT a large retrospective study of 962 MPN-T patients subdivided in 323 367 311 267 226 197 174 142 124 103 87 72 Hetero PV and 639 ET patients [48]. Aspirin responsive platelet thrombophilia 13 12 10 9 9 7 4 4 4 4 2 Homo or microvascular symptoms due to microvessel disorder including Figure 16: Retrospective study on the probability of cardiovascular thrombotic migraine-like headache, acral paresthesia, erythromelalgia, transient event-free survival (days up to 3600 days=10 years) according to the JAK2V617F neurological and visual disturbances (Sticky Platelet Syndome 49) were mutational state in 323 PV and 639 ET patients (Vannucchi et al. [20]). Only excluded by definition and not considered in this retrospective analysis major thrombotic events were retrospectively recorded not including the erythromelalgic peripheral and cerebral ischemic events [37]. The overall [48]. Major thrombotic events were assessed as could be objectively incidence of major thrombotic events in JAK2V617F mutated PV patients during documented in the medical records including ischemic stroke, transient 10 years follow-up is about 25%, a similar incidence of thrombotic events as ischemic attacks, myocardial infarction, angina pectoris, deep vein in the study of Messinezy et al in PV not using aspirin [48]. Source Alessandro M. Vannucchi et al. [48]. thrombosis abdominal vein thrombosis, and pulmonary embolism

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PV: WHO-ECMP stage 0 1 2 3 4 5 6 WHO-ECMP Prodromal Erythrocy-themic Overt PV PV PMF Post-PV MF Leukemic PV Early PV Clinical Diagnosis masked PV PV Classical PV Masked PV Spent PV MDS/AL LAP-score ↑ ↑ ↑ ↑ ↑/↑↑ variable variable Red cell mass (RCM) N ↑ ↑ ↑ ↑ variable N/↓ Serum EPO N/↓ N/↓ ↓ ↓ ↓ variable N/↓ Erythrocytes x1012/l >5.8 <5.8 >5.8 >5.8 >5.8 variable N/↓ Leukocytes x109/l <12 <12 <15 < or->15 >15 >20 >20 Platelets x109/l >400 ,400 < or >400 >400 < or >1000 variable variable WHO-ECMP bone marrow Early PV Early PV Early PV Trilinear PV Trilinear PV Myelofibrosis Leukemic Bone marrow cellularity (%) 50-80 50-80 60-100 80-100 80-100 Decreased Increased Grading reticulin fibrosis: RF RF 0-1 RF 0-1 RF 0-1 RF 0/1, RCF 2/3 RCF 3/ 4 Grading myelofibrosis: MF57 MF 0 MF 0 MF 0 MF 0 MF 1/2 MF 2/3 Splenomegaly on palpation No/+ No No/+ + ++/+++ /large large Spleen size, echogram cm <12-15 <13 15-Dec 18-Dec 18->20 >20 >20 Spleen size on palpation cm 0-3 NP 0-3 6-Apr >6 >8 >8 JAK2V617F in Granulocytes % low low Moderate <50 High >50 High >50 High >50 No or ++ JAK2V617F in BFU-e (exon 12) +(++) +(++) +(++) ++ ++ ++ Therapeutic implications Low risk Low risk Low risk Intermediate risk High risk Post-PV MF Leukemia Anno 2014 MPN MPN MPN PV PV-MF Spent phase PV First line Aspirin/Phlebotomy Aspirin Aspirin Phlebotomy Phlebotomy* If IFN resistant ◊ JAK2 Chemotherapy Bone marrow Second line IFN versus Phlebotomy Phlebotomy Aspirin Aspirin HU or JAK2 Inhibitor ◊ transplantation? Low dose IFN Hydroxyurea (HU) IFN resistant ◊ inhibitor Bone marrow Supportive responsive HU Third line JAK2 inhibitor transplantation Table 8: Staging of JAK2V617F positive prodromal PV, erythrocythemic PV, and five stages of PV according to WHO-ECMP criteria related to therapy anno 2013 [41] *↑ = increased, ↓ = decreased, N = normal, + = present or heterozygous; ++ = homozygous. of the literature in 1241 ET patients from 14 retrospective studies caution and withheld as long as phlebotomy and low dose aspirin are [50]. The frequency of bleeding was higher in JAK2V617F homozygous effective in the treatment of early and intermediate plethoric PV stages (21.4%) than in wild type or heterozygous ET patients, 3.1% and 3.8% 1 and 2. In the 1980 French PV study in PV patients under the age respectively. The higher bleeding tendency in homozygous JAK2V617F of 65 years, toxicity of hydroxyurea was observed within 5 years in MPN patients is predicted to be related to higher erythrocyte counts at 29% of 133 PV patients, which was limited to dry skin and acne in 7%, increased platelet and leukocyte counts and its pathophysiology of the gastric pairddiarrhea in 9%, aphthous ulcers in the mouth in lo%, and underlying mechanisms is currently under our investigation. leg ulcers in 9% [56]. Leg ulcers only healed after discontinuation of hydroxyurea; these complications generally appear late within 5 years In the second Vannucchi study, a total of 214 patients were or more after initial treatment. Dry skin in 1, aphthous stomatitis in 4 treated with phlebotomy, 58% of 219 PV and 4% of 257 ET patients. and leg ulcers in 10 cases were the reasons for replacing hydroxyurea by Myelosuppressive chemotherapy was administered to 497 patients pipobroman in 9%. In the update 10 years later in 1997, the incidence (52%) including 59% of 219 PV and 48% of 257 ET patients [48]. For of leukemia was about 10% at 13 years in hydroxyurea treated PV- comparison, in the Dutch 2008 survey of 363 MPN (123 ET, 190 PV and patients and life expectancy was 70% at 14 years as compared to 83.7% 50 MF) patients 93% of PV, 71% of ET and 37% of MF were on aspirin in age-matched controls [56]. The frequency of MF or spent phase PV mainly because of microvascular symptoms including migraine-like in the update in 1997 was 17% at 10 years and 40% at 16 years. The headache, acral paresthesia, erythromelalgia, transient neurological final analysis of this 1980 French PVSG study of HU as the upfront and visual disturbances. Phlebotomy became the first line treatment in first-line therapy in 136 evaluable PV patients younger than 65 years 6% of ET, 78% of PV and 9% of MF when the Dutch recommendations is published in 2011 [57]. The cumulative incidence (probability) of are applied [51,52]. myelofibrosis (MF) at 10, 15 and 20 years was 15%, 24% and 32% in the Because of advanced or symptomatic MPN disease 31% of ET, 29% HU arm and the cumulative incidence of AML/MDS at 10, 15 and 20 of PV and 30% of MF were on treatment with hydroxyurea and 16% years was 7.3, 10.7% and 16.6% for HU treated PV patients. As shown of ET, 16% of PV and 4% of MF were on treatment with pegylated in Table 8, high risk PV with advanced MPN-T disease in ET and PV interferon (PegasysR) [11,54,55]. The 20% difference of HU use patients in terms of high JAK2V617F allele burden, progressive MPN (50% of Italian MPN-T patients versus 30% of Dutch MPN patients) disease, splenomegaly and constitutional symptoms are candidates for can readily be ascribed to significant differences in the Italian IPSET myelosuppressive (hydroxyurea) or myeloreductive (JAK2 inhibtors) recommendations 42 versus the Dutch guidelines for MPN disease in treatment [11,41]. ET and PV patients [50-54]. References Hydroxyurea is Not an Innocent Myelosppresive Agent 1. Mitchell SW (1878) On a rare vaso-motoe neurosis og the extremities and on in PV and ET themaladies with which it may be confounded Ame. J Med Sci 76: 2-36. 2. Smith LA, Allen FV (1938) Erythermalgia (erythromelalgia) of the extremities. Hydroxyurea is not an innocent drug and should be used with A syndrome characterized by redness, heat and pain. Am Heart J 16: 175-188.

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3. Michiels JJ (1997) Erythromelalgia and thrombocythemia: a disease of platelet 25. van Genderen PJ, Budde U, Michiels JJ, van Strik R, van Vliet HH (1996) prostaglandin metabolism--thesis, Rotterdam, 1981. Semin Thromb Hemost The reduction of large von Willebrand factor multimers in plasma in essential 23: 335-338. thrombocythaemia is related to the platelet count. Br J Haematol 93: 962-965.

4. Michiels JJ, Abels J, Steketee J, van Vliet HH, Vuzevski VD (1985) 26. Van Genderen PJJ, Prins FJ, Lucas IS, van de Moesdijk D, van vliet HHDM Erythromelalgia caused by platelet-mediated arteriolar inflammation and et al. (1997) Decreased half-life time of plasma von Willebrand factor collagen thrombosis in thrombocythemia. Ann Intern Med 102: 466-471. binding activity in essential thrombocythemia: normalization after cytoreduction of the increased platelet cout. Br J Haematol 99: 832-836. 5. Michiels JJ, van Joost T (1990) Erythromelalgia and thrombocythemia: a causal relation. J Am Acad Dermatol 22: 107-111. 27. van Genderen PJ, Leenknegt H, Michiels JJ (1997) The paradox of bleeding 6. Michiels JJ, Berneman Z, Schroyens W, van Urk H (2003) Aspirin-responsive and thrombosis in thrombocythemia: is von Willebrand factor the link? Semin painful red, blue, black toe, or finger syndrome in polycythemia vera associated Thromb Hemost 23: 385-389. with thrombocythemia. Ann Hematol 82: 153-159. 28. (1981) Treatment of polycythaemia vera by radiophosphorus or busulphan: 7. Michiels JJ, ten Kate FW, Vuzevski VD, Abels J (1984) Histopathology of a randomized trial. “Leukemia and Hematosarcoma” Cooperative Group, erythromelalgia in thrombocythaemia. Histopathology 8: 669-678. European Organization for Research on Treatment of Cancer (E.O.R.T.C.). Br J Cancer 44: 75-80. 8. van Genderen PJ, Michiels JJ (1997) Erythromelalgia: a pathognomonic microvascular thrombotic complication in essential thrombocythemia and 29. Messinezy M, Pearson TC, Prochazka A, Wetherley-Mein G (1985) Treatment polycythemia vera. Semin Thromb Hemost 23: 357-363. of primary proliferative polycythaemia by venesection and low dose busulphan: retrospective study from one centre. Br J Haematol 61: 657-666. 9. Michiels JJ, Koudstaal PJ, Mulder AH, van Vliet HH (1993) Transient neurologic and ocular manifestations in primary thrombocythemia. Neurology 43: 1107- 30. Van de Pette JE, Prochazka AV, Pearson TC, Singh AK, Dickson ER, et al. 1110. (1986) Primary thrombocythaemia treated with busulphan. Br J Haematol 62: 229-237. 10. Michiels JJ, van Genderen PJ, Lindemans J, van Vliet HH (1996) Erythromelalgic, thrombotic and hemorrhagic manifestations in 50 cases of 31. Michiels JJ (1999) Aspirin and platelet-lowering agents for the prevention thrombocythemia. Leuk Lymphoma 22 Suppl 1: 47-56. of vascular complications in essential thrombocythemia. Clin Appl Thromb Hemost 5: 247-251. 11. Michiels JJ, Ten Kate FWJ, Koudstaal PJ, Van Genderen PJJ (2013) Aspirin responsive platelet thrombophilia in essentialthrombocythemia and 32. Michiels JJ (1999) Normal life expectancy and thrombosis-free survival in polycythemia vera. World J Hematol 2: 20-41. aspirin treated essential thrombocythemia. Clin Appl Thromb Hemost 5: 30-36. 12. Laszlo J (1975) Myeloproliferative disorders (MPD): myelofibrosis, 33. LandolfiR, Marchioli R. Barbui T, Michiels JJ, Najean Y et al. (1997). Treatment myelosclerosis, extramedullary hematopoiesis, undifferentiated MPD, and of polycythemia vera. Sem Thromb Hemostas 23: 473-478. hemorrhagic thrombocythemia. Semin Hematol 12: 409-432. 34. Michiels JJ. (2014) Erythromelalgic Thrombotic Thrombocythemia (ETT) 13. Murphy S, Iland H, Rosenthal D, Laszlo J (1986) Essential thrombocythemia: and Primary Hemorrhagic Trombocythemia (PHT) in patients with Essential an interim report from the Polycythemia Vera Study Group. Semin Hematol 23: Thrombocythaemia (ET) and Polycythaemia Vera (PV). Review of 100 PHT 177-182. and 99 ETT cases from the literature 1908-1980 in view of the 1975 PVSG criteria for ET and PV. JHTD This Issue. 14. Berlin NI (1975) Diagnosis and classification of the polycythemias. Semin Hematol 12: 339-351. 35. van Genderen PJ, Michiels JJ (1994) Erythromelalgic, thrombotic and haemorrhagic manifestations of thrombocythaemia. Presse Med 23: 73-77. 15. Kurnick JE, Ward HP, Block MH (1972) Bone marrow sections in the differential diagnosis of polycythemia. Arch Pathol 94: 489-499. 36. Van Genderen PJ, Michiels JJ (1995) Hydroxyurea in essential thrombocytosis. 16. van Genderen PJ, Michiels JJ, van Strik R, Lindemans J, van Vliet HH (1995) N Engl J Med 333: 802-803. Platelet consumption in thrombocythemia complicated by erythromelalgia: 37. van Genderen PJ, Mulder PG, Waleboer M, van de Moesdijk D, Michiels JJ reversal by aspirin. Thromb Haemost 73: 210-214. (1997) Prevention and treatment of thrombotic complications in essential 17. Michiels JJ, Zijlstra FJ (1993) Prostaglandin cyclooxygenase products but thrombocythaemia: efficacy and safety of aspirin. Br J Haematol 97: 179-184. not thromboxane A(2) are involved in the pathogenesis of ewthromelalgia in 38. Michiels JJ (1997) Erythromelalgia and vascular complications in polycythemia thrombocythaemia. Mediators Inflamm 2: 385-389. vera. Semin Thromb Hemost 23: 441-454. 18. van Genderen PJ, Prins FJ, Michiels JJ, Schrör K (1999) Thromboxane- 39. Landolfi R, Marchioli R, Kutti J, Gisslinger H, Tognoni G, et al. (2004) Efficacy dependent platelet activation in vivo precedes arterial thrombosis in and safety of low-dose aspirin in polycythemia vera. N Engl J Med 350: 114- thrombocythaemia: a rationale for the use of low-dose aspirin as an 124. antithrombotic agent. Br J Haematol 104: 438-441. 40. Cortelazzo S, Viero P, Finazzi G, D’Emilio A, Rodeghiero F, et al. (1990) 19. Van Genderen PJJ, Lucas IS, van Strik R, Vuzevski VD, Prins F et al. (1996) Incidence and risk factors for thrombotic complications in a historical cohort of Erythromelalgia in essential thrombocythemia is characterized by platelet 100 patients with essential thrombocythemia. J Clin Oncol 8: 556-562. activation and endothelial cell damage but not by thrombin generation, Thromb Haemostas 76: 333-338. 41. Michiels JJ, Ten Kate FWJ, Koudstaal PF, Van Genderen PJJ (2013)Aspirin responsisive platelet thrombophilia in essential thrombocythemiaand 20. Michiels JJ, Berneman Z, Schroyens W, Finazzi G, Budde U et al. (2006). polycythemia vera. World J Hematol 2: 20-43 The paradox of platelet activation and impaired function; platelet-von Willebrand factor interactions and the etiology of thrombotic and hemorrhagic 42. Passamonti F, Thiele J, Girodon F,Rumi E,Carobbio A et al. (2012) A prognostic manifestations in essential thrombocythemia and polycythemia vera. Sem model to predict survival in 867 WHO-defined essential thrombocythemia at Thromb Hemostas 32: 589-604. diagnosis. Blood 120: 1197-1201. 21. Budde U, Dent JA, Berkowitz SD, Ruggeri ZM, Zimmerman TS (1986) 43. Cortelazzo S, Finazzi G, Ruggeri M, Vestri O, Galli M, et al. (1995) Hydroxyurea Subunit composition of plasma von Willebrand factor in patients with the for patients with essential thrombocythemia and a high risk of thrombosis. N myeloproliferative syndrome. Blood 68: 1213-1217. Engl J Med 332: 1132-1136.

22. Budde U, Scharf RE, Franke P, Hartmann-Budde K, Dent J, et al. (1993) 44. Scott LM, Scott MA, Campbell PJ, Green AR (2006) Progenitors homozygous Elevated platelet count as a cause of abnormal von Willebrand factor multimer for the V617F mutation occur in most patients with polycythemia vera, but not distribution in plasma. Blood 82: 1749-1757. essential thrombocythemia. Blood 108: 2435-2437.

23. van Genderen PJ, Michiels JJ, van der Poel-van de Luytgaarde SC, van 45. Moliterno AR, Williams DM, Rogers O, Isaacs MA, Spivak JL (2008) Phenotypic Vliet HH (1994) Acquired von Willebrand disease as a cause of recurrent variability within the JAK2 V617F-positive MPD: roles of progenitor cell and mucocutaneous bleeding in primary thrombocythemia: relationship with platelet neutrophil allele burdens. Exp Hematol 36: 1480-1486. count. Ann Hematol 69: 81-84. 46. Godfrey AL, Chen E, Pagano F, Ortmann CA, Silber Y et al. (2012) JAK2V617F 24. van Genderen PJ, Leenknegt H, Michiels JJ, Budde U (1996) Acquired von homozygosity arises commonly and recurrently in PV and ET, butPV is Willebrand disease in myeloproliferative disorders. Leuk Lymphoma 22 Suppl characterized by expansion of a dominant homozygous subclone. Blood 120: 1: 79-82. 2704-2707

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47. Vannucchi AM, Antonioli E, Guglielmelli P, Longo G, Pancrazzi A, et al. (2007) 53. Michiels JJ Schouten HC (2006) Artsenbrochure Myeloproliferative Disorders Prospective identification of high-risk polycythemia vera patients based on (MPD) Essentiaelel. Thrombocythemia, Polycythemia Vera Chronische JAK2(V617F) allele burden. Leukemia 21: 1952-1959. Idiopathische Myelofibrose. Nederlandse MPD Stichting.

48. Vannucchi AM, Antonioli E, Guglielmelli P, Rambaldi A, Barosi G, et al. 54. Commendeur S, Michiels JJ, te Boekhorst PAW, Schouten HC, Zweegman S (2007) Clinical profile of homozygous JAK2 617V>F mutation in patients with (2008) Quality of life, social activity and work participation of MPD patients in polycythemia vera or essential thrombocythemia. Blood 110: 840-846. The Netherlands: a survey of 363 MPD patients. The Dutch MPD Foundation.

49. Kubisz P, Stasko J, Holly P (2013) Sticky platelet syndrome. Semin Thromb 55. Kiladjian JJ, Cassinat B, Turlure P, Cambier N, Roussel M (2008) High Hemost 39: 674-683. molecular response rate of polycythemia vera treated with peglyated interferon- alpha-2a. Blood 108: 1281. 50. Griesshammer M1, Bangerter M, van Vliet HH, Michiels JJ (1997) Aspirin in essential thrombocythemia: status quo and quo vadis. Semin Thromb Hemost 56. Najean Y, Rain JD (1997) Treatment of polycythemia vera: the use of 23: 371-377. hydroxyurea and pipobroman in 292 patients under the age of 65 years. Blood 90: 3370-3377. 51. Michiels JJ, Barbui T, Finazzi G, Fuchtman SM, Kutti J, et al. (2000) Diagnosis and treatment of polycythemia vera and possible future study designs of the 57. Kiladjian JJ, Chevret S, Dosquet C, Chomienne C, Rain JD (2011) Treatment PVSG. Leuk Lymphoma 36: 239-253. of polycythemia vera with hydroxyurea and pipobroman: final results of a randomized trial initiated in 1980. J Clin Oncol 29: 3907-3913. 52. Michiels JJ, De Raeve H, Hebeda K, Lam KH, Berneman Z, et al. (2007) WHO bone marrow features and European clinical, molecular, and pathological (ECMP) criteria for the diagnosis of myeloproliferative disorders. Leuk Res 31: 1031-1038.

J Hematol Thromb Dis ISSN: 2329-8790 JHTD, an open access journal Volume 2 • Issue 5 • 1000149